1. Field of Invention
The present inventive subject matter relates to novel methods for detecting the presence of a biological substance of interest in a test sample of saliva, oral or other bodily fluids. Also provided is a miniaturized, portable, hand-held apparatus for measuring the fluorescence polarization of a liquid sample.
2. Description of the Related Art
Immunoassays. Infectious disease rates and immunization strategies continue to evolve in the United States and worldwide in response to societal needs, national defense, and evolutionary changes in the organisms producing disease. Immunizations are performed to prevent many infections, while prophylactic population screening is utilized for infections lacking effective vaccines and for those diseases having a low enough incidence that mass immunization is not deemed most efficacious.
The current method for diagnosis of disease, determining exposure to biological materials such as pathogens, or monitoring immunization status varies depending on the specific assay. Some methods employ an in vivo assay. Others require a biological sample, such as blood or serum, to be obtained and tested. Tests performed usually are one of the non-homogeneous type diagnostic methods such as enzyme-linked immunosorbant assay (hereinafter “ELISA”), radioimmunoassay (hereinafter “RIA”), or agglutination. All are surface-binding, heterogeneous assays and require the antigen of interest to interact with a surface to achieve success, often at the expense of high non-specific binding and loss of specificity.
Fluorescence Polarization
Fluorescence polarization (hereinafter “FP”) is the process in which visible or ultraviolet light is polarized with a filter and shines on part of a molecule, the fluorochrome, that in turn fluoresces, emitting light of longer wavelength whose signal is captured and recorded. The emitted light recorded stays more polarized in solutions when there are slower turning, large molecule-fluorochrome complexes than when there are smaller labeled molecules. Different fluorochromes can be chosen to accommodate molecules of different sizes up to 107 kDa molecular weight.
U.S. Pat. No. 4,585,862 to Wang, et al., issued Apr. 29, 1986, discloses a method and reagents for determining a ligand, particularly steroid, hormone, antiasthmatic, antineoplastic, antiarrhythmic, anticonvulsant, antibiotic, antiarthritic, antidepressant, cardiac glycoside, or a metabolite thereof, in biological fluids such as serum, plasma, spinal fluid, amnionic fluid, and urine. In particular, Wang, et al. relates to a specific class of tracer compounds required as reagents in such procedures.
U.S. Pat. No. 5,976,820 to Jolley, et al., issued Nov. 2, 1999, discloses a homogeneous immunoassay in which a fluorophore-conjugated lipopolysaccharide derived bacterial antigen is reacted with antibodies specific for the antigens in a diluted serum specimen, with quantitative detection of the formation of an immune complex obtained by measuring the change in fluorescence polarization after complex formation.
U.S. Pat. No. 6,432,632 to Nakayama, et al., issued Aug. 13, 2002, discloses a fluorescence polarization method for analyzing an assay-object in a sample, comprising the steps of: (a) providing a fluorescent-labeled protein in which a protein is covalently bound to a fluorochrome(s), wherein the protein is capable of specifically binding to the assay-object; (b) allowing the fluorescent-labeled protein to bind to the assay-object; and (c) measuring a change in the degree of fluorescence polarization which has taken place in the fluorescent-labeled protein by its binding to the assay-object.
Saliva and Oral Fluids
Saliva and oral fluids are biochemically distinct and have been increasingly recognized as acceptable alternatives to serum for use in diagnostic tests for certain hormones, drugs, antibodies and antigens. Oral fluids are collected without pain, needle sticks, or religious and social prohibitions, and their use involves minimal risk or exempt protocols for the use of human subjects. Surprisingly, diagnostic assays utilizing saliva and other oral fluids appear as entries in the National Library of Medicine MEDLARS database with a frequency of only 1 in 30 and 1 in 100, respectively, when compared to entries in which blood serum is reported.
Knowledge of the epidemiology of diseases is important in health care planning and treatment. Both are dependent upon accurate and rapid diagnosis. Using an under-utilized and elegant technology, fluorescence polarization, we have developed assays for accurate and rapid detection and diagnosis of antibodies to pathogens producing diseases of military and commercial interest. The technology is homogeneous and can accurately estimate concentrations of diagnostic markers, drugs and chemicals, or bio-hazardous agents in oral fluids and environmental samples within a few seconds to several minutes. Further, the inventive oral immunodiagnostic assay systems are robust and relatively unaffected by use with non-homogeneous samples, such as whole blood or saliva. Specificity of FP assays is generally very high, approximately 98%, with good sensitivity. The applications of FP salivary diagnostics to determination of tuberculosis exposure and of anthrax immunization status are provided as examples.
There is potential for widespread use of these assays as non-invasive tests, especially as more compact, simplified fluorescence polarimeters become available. FP tests are provided and are applicable to a wide spectrum of microorganisms. Rapid FP salivary diagnostics, have both military and civilian applications and may be used in a clinic or field setting to aid in diagnosis of disease, detection of exposure, or verification of vaccination. Particularly in those instances where rapid detection in field situations is required, these considerations and continuing improvements in standardization of collection methods make the use of saliva and oral fluids as well as bodily fluids a novel and effective diagnostic media of choice for the future.
Fluorescence Polarization Detection Instrumentation
Fluorescence polarization instruments and their use for clinical applications are described, for example, in “Design, Construction, and Two Applications for an Automated Flow-Cell Polarization Fluorometer with Digital Read Out”; R. D. Spencer, F. D. Toledo, B. T. Williams, and N. L. Yoss; Clinical Chemistry, 19/8, pages 838-344 (1973). Such instruments can rapidly analyze body fluid samples labeled with a fluorescent material.
Early instruments of such type required relatively high wattage, intense light sources, such as 200-250 watt mercury or xenon gas discharge lamps, in order to obtain the desired emission radiation from the sample at a signal to noise level ratio sufficient for detection and amplification by a low-noise photomultiplier tube and associated electronics. Such high wattage, bright lamps also require substantial cooling in order to maintain the integrity of the optical system.
An improved fluorescence polarization instrument is described in U.S. Pat. No. 4,516,856, issued to Popelka on May 14, 1985. Popelka discloses an optical system for a fluorescent polarization instrument including a low wattage, low intensity focused light source and a polarizer/liquid crystal combination in the excitation path focusing excitation light of alternate planes of perpendicular polarization onto a fluorescent liquid sample, wherein emitted light from the fluorescent sample is filtered, polarized and focused onto a photomultiplier for processing, and a series of non-reflective baffles are placed around the sample to reduce reflections; monitoring means monitor the excitation light and maintain a substantially constant intensity level focused on the sample, while the low wattage, low intensity light source is provided by a 50 watt tungsten halogen projector lamp. An exemplary prior art instrument schematic is shown in FIG. 1.
Even with these improvements, current instruments are large, bulky tabletop instruments with high voltage and high power usage, and thus substantial AC power requirements. Further, current FP instruments suffer from intrinsic background fluorescence contributed by fluorescent biological molecules such as NAD, NADH, NADP, and NADPH. In addition, current FP instruments also suffer from intrinsic AC line voltage noise. Thus, there is a need for improved fluorescence polarization instruments which are smaller, lighter, and operate using a battery pack or other low power DC power source; avoid the intrinsic background fluorescence contributed by fluorescent biological molecules such as NAD, NADH, NADP, and NADPH; and offer a simplified optics block design. In particular, with increasing prevalence of non-invasive FP tests applicable to a wide spectrum of microorganisms, to aid in diagnosis of disease, detection of exposure, or verification of vaccination, both military and civilian applications in a clinic or field setting will require portable FP instruments. The inventive apparatus satisfies this need by providing a miniaturized, portable apparatus for measuring the fluorescence polarization of a liquid sample.